Expression of lipoproteins

ABSTRACT

Heterologous lipidated proteins formed recombinantly are disclosed and claimed. The expression system can be  E. coli.  The heterologous lipidated protein has a leader sequence which does not naturally occur with the protein portion of the lipidated protein. The lipidated protein can have the  Borrelia  OspA leader sequence. The protein portion can be OspC, PspA, UreA, Ure B, or a fragment thereof. Methods and compositions for forming and employing the proteins are also disclosed and claimed.

REFERENCE TO RELATED APPLICATIONS

Reference, especially with respect to recombinant Borrelia proteins, ismade to each of applications Ser. No. 07/973,338; filed Oct. 29, 1992;Ser. No. 08/373,455 (Rule 62 FWC of U.S. Ser. No. 07/973,338), filedJan. 17, 1995, Ser. No. 07/888,765, filed May 27, 1992; Ser. No.08/211,891; filed Oct. 16, 1992 (national phase of PCT/US92/08697); andSer. No. 07/779,048, filed Oct. 18, 1991.

Reference, especially with respect to structural genes of pneumococcalproteins, epitopic regions thereof, and administration of pneumococcalproteins, is made to each of applications Ser. Nos. 656,773, filed Feb.15, 1991; Ser. No. 835,698, filed Feb. 12, 1992; Ser. No. 072,056, filedJun. 3, 1993; Ser. No. 072,068, filed Jun. 3, 1993; Ser. No. 214,222,filed Mar. 17, 1994; Ser. No. 214,164, filed Mar. 17, 1994; Ser. No.247,491, filed May 23, 1994; Ser. No. 048,896, filed Apr. 20, 1993; Ser.No. 246,636, filed May 20, 1994; ______ (continuation-in-part ofapplication Ser. No. 246,636), filed October 7, 1994; ______, filed Jun.2, 1995 (Attorney Docket No. 454312-2040, Briles et al., entitled, “ORALADMINISTRATION OF PNEUMOCOCCAL ANTIGENS”); ______, filed May 19, 1995(Attorney Docket No. 454312-2018); Ser. No. 08/312,949, filed Sep. 30,1994; and _____, filed Jun. 7, 1995 (Attorney Docket No. 454312-2051,Becker et al.,entitled, “IMMUNOGENIC COMBINATION COMPOSITIONS ANDMETHODS”).

Reference is also made to application Ser. No. ______, filed Jun. 7,1995 (Attorney Docket No. 454312-2052, Huebner et al., entitled,“EXPRESSION OF LIPOPROTEINS”).

Each of the aforementioned applications is hereby incorporated herein byreference.

FIELD OF INVENTION

The present invention is concerned with genetic engineering to effectexpression of lipoproteins from vectors containing nucleic acidmolecules encoding the lipoproteins. More particularly, the presentinvention relates to expression of a recombinant lipoprotein wherein thelipidation thereof is from expression of a first nucleic acid sequenceand the protein thereof is from expression of a second nucleic acidsequence, the first and second nucleic acid sequences, which do notnaturally occur together, being contiguous. The invention furtherrelates to expression of such lipoproteins wherein the first nucleicacid sequence encodes a Borrelia lipoprotein leader sequence. Theinvention also relates to recombinant lipidated proteins expressed usingthe nucleic acid sequence encoding the OspA leader sequence, methods ofmaking and using the same compositions thereof and methods of using thecompositions. The invention additionally relates to nucleic acidsequences encoding the recombinant lipoproteins, vectors containingand/or expressing the sequences, methods for expressing the lipoproteinsand methods for making the nucleic acid sequences and vectors;compositions employing the lipoproteins, including immunogenic orvaccine compositions, such compositions preferably having improvedimmunogenicity; and methods of using such compositions to elicit animmunological or protective response.

Throughout this specification, various documents are referred to inorder to more fully describe the state of the art to which thisinvention pertains. These documents are each hereby incorporated hereinby reference.

BACKGROUND OF THE INVENTION

Lyme borreliosis is the most prevalent tick-borne disease in the UnitedStates as well as one of the most important tick-borne infectiousdiseases worldwide. The spirochete Borrelia burgdorferi is the causativeagent for Lyme disease. Infection with B. burgdorferi produces local andsystemic manifestations. Local symptoms that appear early afterinfection are a skin lesion at the site of the tick bite, termederythema migrans. Weeks to months after infection, systemicmanifestations that include rheumatic, cardiac and neurological symptomsappear. The early local phase of B. burgdorferi infection is easilytreatable with antibiotics. However, the later systemic phases haveproved to be more refractory to antibiotics.

Substantial effort has been directed toward the development of a vaccinefor Lyme disease. Two distinct approaches have been used for vaccinedevelopment. One approach is to use a vaccine composed of wholeinactivated spirochetes, as described by Johnson in U.S. Pat. No.4,721,617. A whole inactivated vaccine has been shown to protecthamsters from challenge and has been licensed for use in dogs.

Due to the concerns about cross-reactive antigens within a whole cellpreparation, human vaccine research has focused on the identificationand development of non-cross-reactive protective antigens expressed byB. burgdorferi. Several candidate antigens have been identified to date.Much of this effort has focused on the most abundant outer surfaceprotein of B. burgdorferi, namely outer surface protein A (OspA), asdescribed in published PCT patent application WO 92/14488, assigned tothe assignee hereof. Several versions of this protein have been shown toinduce protective immunity in mouse, hamster and dog challenge studies.Clinical trials in humans have shown the formulations of OspA to be safeand immunogenic in humans [Keller et al., JAMA (1994) 271:1764-1768].Indeed, one formulation containing recombinant lipidated OspA asdescribed in the aforementioned WO 92/14488, is now undergoing Phase IIIsafety/efficacy trials in humans.

While OspA is expressed in the vast majority of clinical isolates of B.burgdorferi from North America, a different picture has emerged fromexamination of the clinical Borrelia isolates in Europe. In Europe, Lymedisease is caused by three genospecies of Borrelia, namely B.burgdorferi, B. garinii and B. afzelli. In approximately half of theEuropean isolates, OspA is not the most abundant outer surface protein.A second outer surface protein C ((OspC) is the major surface antigenfound on these spirochetes. In fact, a number of European clinicalisolates that do not express OspA have been identified. Immunization ofgerbils and mice with purified recombinant OspC produces protectiveimmunity to B. burgdorferi strains expressing the homologous OspCprotein [V. Preac-Mursic et al., INFECTION (1992) 20:342-349; W. S.Probert et al., INFECTION AND IMMUNITY (1994) 62:1920-1926]. The OspCprotein is currently being considered as a possible component of asecond generation Lyme vaccine formulation.

Recombinant proteins are promising vaccine or immunogenic compositioncandidates, because they can be produced at high yield and purity andmanipulated to maximize desirable activities and minimize undesirableones. However, because they can be poorly immunogenic, methods toenhance the immune response to recombinant proteins are important in thedevelopment of vaccines or immunogenic compositions.

A very promising immune stimulator is the lipid moietyN-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy)propyl-cysteine, abbreviatedPam₃Cys. This moiety is found at the amino terminus of the bacteriallipoproteins which are synthesized with a signal sequence that specifieslipid attachment and cleavage by signal peptidase II. Synthetic peptidesthat by themselves are not immunogenic induce a strong antibody responsewhen covalently coupled to Pam₃Cys [Bessler et al., Research Immunology(1992) 143:548-552].

In addition to an antibody response, one often needs to induce acellular immune response, particularly cytoxic T lymphocytes (CTLB).Pam₃Cys-coupled synthetic peptides are extremely potent inducers ofCTLs, but no one as yet reported CTL induction by large recombinantlipoproteins.

The nucleic acid sequence and encoded amino acid sequence for OspA areknown for several B. burgdorferi clinical isolates and is described, forexample, in published PCT application WO 90/04411 (Symbicom AB) for B31strain of B. burgdorferi and in Johnson et al., Infect. Immun.60:1845-1853 for a comparison of the ospA operons of three B.burgdorferi isolates of different geographic origins, namely B31, ACA1and Ip90.

As described in WO 90/04411, an analysis of the DNA sequence for the B31strain shows that the OspA is encoded by an open reading frame of 819nucleotides starting at position 151 of the DNA sequence and terminatingat position 970 of the DNA sequence (see FIG. 1 therein). The firstsixteen amino acid residues of OspA constitute a hydrophobic signalsequence of OspA. The primary translation product of the full length B.burgdorferi gene contains a hydrophobic N-terminal signal sequence whichis a substrate for the attachment of a diacyl glycerol to the sulfhydrylside chain of the adjacent cysteine residue. Following this attachment,cleavage by signal peptidase II and the attachment of a third fatty acidto the N-terminus occurs. The complete lipid moiety is termed Pam₃Cys.It has been shown that lipidation of OspA is necessary forimmunogenicity, since OspA lipoprotein with an N-terminal Pam₃Cys moietystimulated a strong antibody response, while OspA lacking the attachedlipid did not induce any detectable antibodies [Erdile et al., Infect.Immun., (1993), 61:81-90].

Published international patent application WO 91/09870 (MikrogenMolekularbiologische Entwicklungs-GmbH) describes the DNA sequence ofthe ospC gene of B. burgdorferi strain Pko and the OspC (termed pC inthis reference) protein encoded thereby of 22 kDa molecular weight. Thissequence reveals that OspC is a lipoprotein that employs a signalsequence similar to that used for OspA. Based on the findings regardingOspA, one might expect that lipidation of recombinant OspC would beuseful to enhance its immunogenicity; but, as discussed below, theapplicants experienced difficulties in obtaining detectable expressionof recombinant OspC.

U.S. Pat. No. 4,624,926 to Inouye relates to plasmid cloning vectors,including a DNA sequence coding for a desired polypeptide linked withone or more functional fragments derived from an outer membranelipoprotein gene of a gram negative bacterium. The polypeptide expressedby thee transformed E. coli host cells comprises the signal peptide ofthe lipoprotein, followed by the first eight amino acid residues of thelipoprotein, which in turn are followed by the amino acid sequence ofthe desired protein. The signal peptide may then be translocatednaturally across the cytoplasmic membrane and the first eight aminoacids of the lipoprotein may then be processed further and inserted intothe outer membrane of the cell in a manner analogous to the normalinsertion of the lipoprotein into the outer membrane. Immunogenicity ofthe expressed proteins was not demonstrated. Moreover, Inouye was not atall concerned with recombinant lipidation, particularly to enhanceimmunogenicity.

Published international patent application WO91/09952 describes plasmidsfor expressing lipidated proteins. Such plasmids involve a DNA sequenceencloding a lipoprotein signal peptide linked to the codons for one ofthe β-turn tetrapeptides QANY or IEGR, which in turn is linked to theDNA sequence encoding the desired protein. Again, immunogenicity of theexpressed proteins was not demonstrated.

Streptoccus pneumoniae causes more fatal infections world-wide thanalmost any other pathogen. In the U.S.A., deaths caused by S. pneumoniaerival in numbers those caused by AIDS. Most fatal pneumoccal infectionsin the U.S.A. occur in individuals over 65 years of age, in whom S.pneumoniae is the most common cause of community-acquired pneumonia. Inthe developed world, most pneumococcal deaths occur in the elderly, orin immunodeficient patents including those with sickle cell disease. Inthe less-developed areas of the world, pneumococcal infection is one ofthe largest causes of death among children less than 5 years of age. Theincrease in the frequency of multiple antibiotic resistance amongpneumococci and the prohibitive cost of drug treatment in poor countriesmake the present prospect for control of pneumococcal diseaseproblematical.

The reservoir of pneumococci that infect man is maintained primarily vianasopharyngeal human carriage. Humans acquire pneumococci first throughaerosols or by direct contact. Pneumococci first colonize the upperairways and can remain in nasal mucosa for weeks or months. As many as50% or more of young children and the elderly are colonized. In mostcases, this colonization results in no apparent infection. In someindividuals, however, the organism carried in the nasopharynx can giverise to symptomatic sinusitis of middle ear infection. If pneumococciare aspirated into the lung, especially with food particles or mucus,they can cause pneumonia. Infections at these sites generally shed somepneumococci into the blood where they can lead to sepsis, especially ifthey continue to be shed in large numbers from the original focus ofinfection. Pneumococci in the blood can reach the brain where they cancause menigitis. Although pneumococcal meningitis is less common thanother infections caused by these bacteria, it is particularlydevastating; some 10% of patients die and greater than 50% of theremainder have life-long neurological sequelae.

In elderly adults, the present 23-valent capsular polysaccharide vaccineis about 60% effective against invasive pneumococcal disease withstrains of the capsular types included in the vaccine. The 23-valentvaccine is not effective in children less than 2 years of age because oftheir inability to make adequate responses to most polysaccharides.Improved vaccines that can protect children and adults against invasiveinfections with pneumococci would help reduce some of the mostdeleterious aspects of this disease.

The S. pneumoniae cell surface protein PspA has been demonstrated to bea virulence factor and a protective antigen. In published internationalpatent application WO 92/14488, there are described the DNA sequencesfor the pspA gene from S. pneumoniae R×1, the production of a truncatedform of PspA by genetic engineering, and the demonstration that suchtruncated form of PspA confers protection in mice to challenge with livepneumococci.

In an effort to develop a vaccine or immunogenic composition based onPspA, PspA has been recombinantly expressed in E. coli. It has beenfound that in order to efficiently express PspA, it is useful totruncate the mature PspA molecule of the R×1 strain from its normallength of 589 amino acids to that of 314 amino acids comprising aminoacids 1 to 314. This region of the PspA molecule contains most, if notall, of the protective epitopes of PspA. However, immunogenicity andprotection studies in mice have demonstrated-that the truncatedrecombinant form of PspA is not immunogenic in naive mice. Thus, itwould be useful to improve the immunogenicity of recombinant PspA andfragments thereof.

Many bacterial and viral pathogens, such as S. pneumoniae andHelicobacter pylori, and HIV, herpes and papilloma viruses gain entrythrough mucosal surfaces. The principal determinant of specific immunityat mucosal surfaces is secretory IgA (S—IgA) which is physiologicallyand functionally separate from the components of the circulatory immunesystem. Mucosal S—IgA responses are predominantly generated by thecommon mucosal immune system (CMIS) [Mestecky, J. Clin. Immunol. (1987),7:265-276], in which immunogens are taken up by specializedlympho-epithelial structures collectively referred to asmucosa-associated lymphoid tissue (MALT). The term common mucosal immunesystem referes to the fact that immunization at any mucosal site canelicit an immune response at all other mucosal sites. Thus, immunizationin the gut can elicit mucosal immunity in the upper airways and viceversa. Further, it is important to note that oral immunization caninduce an antigen-specific IgG response in the systemic compartment inaddition to mucosal IgA antibodies [McGhee, J. R. et al., (1993),Infect. Agents and Disease 2:55-73].

Most soluble and non-replicating antigens are poor mucosal immunogens,especially by the peroral route, probably because they are degraded bydigestive enzymes and have little or no tropism for the gut associatedlymphoid tissue (GALT). Thus, a method for producing effective mucosalimmunogens, and vaccines and immunogenic compositions containing them,would be desirable.

Of particular interest is H. pylori, the spiral bacterium whichselectively colonizes human gastric mucin-secreting cells and is thecausative agent in most cases of nonerosive gastritis in humans. Recentresearch indicates that H. pylori, which has a high urease activity, isresponsible for most peptic ulcers as well as many gastric cancers. Manystudies have suggested that urease, a complex of the products of theureA and ureB genes, may be a protective antigen, However, until now ithas not been known how to produce a sufficient-mucosal immune responseto urease.

Antigens, such as OspC, PspA, UreA, UreB or immunogenic fragmentsthereof, stimulate an immune response when administered to a host. Suchantigens, especially when recombinantly produced, may elicit a strongerresponse when administered in conjunction with an adjuvant. Currently,alum is the only adjuvant licensed for human use, although hundreds ofexperimental adjuvants such as cholera toxin B are being tested.However, these adjuvants have deficiencies. For instance, while choleratoxin B is not toxic in the sense of causing cholera, there is generalunease about administering a toxin associated with a disease as harmfulas cholera, especially if there is even the most remote chance of minorimpurity.

Thus, it would be desirable to enhance the immunogenicity of antigens,by methods other than the use of an adjuvant, especially in monovalentpreparations; and, in multivalent preparations, to have the ability toemploy such a means for enhanced immunogenicity with an adjuvant, so asto obtain an even greater immunological response.

As to expression of recombinant proteins, it is expected that theskilled artisan is familiar with the various vector systems availablefor such expression, e.g., bacteria such as E. coli and the like.

It is believed that heretofore the art has not taught or suggested:expression of a recombinant lipoprotein wherein the lipidation thereofis from expression of a first nucleic acid sequence, the protein thereofis from expression of a second nucleic acid sequence, the first andsecond sequences, which do not naturally occur together, beingcontiguous, especially such a lipoprotein wherein the first sequenceencodes a Borrelia lipoprotein leader sequence, preferably an OspAleader sequence, and even more preferably wherein the first sequenceencodes an OspA leader sequence and the second sequence encodes OspC,PspA, UreA, UreB, or an immunogenic fragment thereof; or genescontaining such sequences; or vectors containing such sequences; ormethods for such expression; or such recombinant lipoproteins; orcompositions containing such recombinant lipoproteins; or methods forusing such compositions; or methods for enhancing the immunogenicity ofa protein by lipidation from a nucleic acid sequence not naturallyoccurring with the nucleic acid sequence encoding the protein portion ofthe lipoprotein.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the invention to provide a recombinant lipoproteinwherein the lipidation thereof is from expression of a first nucleicacid sequence and the protein portion thereof is from expression of asecond nucleic acid sequence and the first and second sequences do notnaturally occur together; especially such a lipoprotein wherein thefirst sequence encodes a Borrelia lipoprotein leader sequence,preferably an OspA leader sequence, and more preferably wherein thesecond sequence encodes a protein portion comprising OspC, PspA, UreA,UreB, or an immunogenic fragment thereof.

It is another object of the invention to provide expression of genesand/or sequences encoding such a recombinant lipoprotein, vectorstherefor and methods for effecting such expression.

It is a further object of the invention to provide immunogeniccompositions, including vaccines, containing the recombinantlipoproteins and/or vectors for expression thereof.

It has surprisingly been found that an immunogenic recombinant lipidatedprotein, preferably OspC or a portion thereof, can be expressed from avector system, preferably E. coli, without the toxicity to the vectorsystem evident when the native lipoprotein signal sequence encodingregion is present. This result has been achieved by replacing thenucleotide sequence encoding the native leader or signal sequence of alipoprotein with the nucleotide sequence encoding a leader or signal ofanother lipoprotein, preferably of a Borrelia lipoprotein, and morepreferably the OspA leader or signal sequence. Proteins not naturallylipidated, such as PspA, UreA, and UreB, may be expressed as recombinantlipidated proteins as well, by fusing the lipoprotein signal sequence tothe first amino acid of the desired protein. These recombinant lipidatedproteins have been shown to elicit an immune response, including amucosal immune response.

It is surprising that fusion of DNA encoding a lipoprotein leadersequence, directly to the DNA encoding a protein, without anyintervening nucleotide sequences, can lead to expression of animmunogenic recombinant lipoprotein in significant quantities withoutthe toxicity evident with the native leader sequence, because previousattempts to express recombinant lipidated proteins have beenunsuccessful. For example, Fuchs et al. report that recombinantly formedOspC (referred to as pC in this reference) with its native leaderprotein was only weakly expressed in E. coli [Mol. Microbiology (1992)6(4):503-509]. Applicants, in addition to Fuchs, attempted to obtainlipidated recombinant OspC by expression of the OspC-encoding sequencein E. coil using the pET vector system described in the aforementionedWO 92/14488 for the expression of OspA and using the pDS12 plasmidsystems described. However, OspC was barely detectable by immunoblottingof cell extracts using these systems to express OspC.

Further, as discussed supra, it was believed that an additionalnucleotide sequence, preferably one encoding a peptide sequence forminga β-turn, was necessary for expression of recombinant lipoproteins, andthe immunogenicity of recombinant lipoproteins previously expressed hadnot been demonstrated.

The procedure of the present invention, therefore, enables largequantities of pure recombinant, immunogenic lipidated proteins, e.g.,OspC, PspA, UreA, UreB and portions thereof, to be obtained, which hasnot heretofore been possible. The recombinantly-formed lipidatedproteins provided herein are significantly more immunogenic than anon-lipidated recombinant analog.

The present invention, it is believed, represents the first instance ofeffecting expression of a heterologous lipidated protein using anon-native, preferably Borrelia and more preferably the ospA leadersequence. The invention, therefore, includes the use of non-native,preferably Borrelia and more preferably the OspA leader sequence toexpress proteins heterologous to the leader sequence.

Accordingly, in one embodiment, the present invention provides anisolated hybrid nucleic acid molecule, preferably DNA, comprising afirst nucleic acid sequence encoding the signal sequence preferably ofan OspA protein of a Borrelia species, coupled in translational openreading frame relationship with a second nucleic acid sequence encodinga mature protein heterologous to the signal sequence, preferably to OspCor PspA. More preferably, the first and second sequences are contiguouswhen the mature protein is naturally lipidated, and separated by onecodon coding for one amino acid, preferably cysteine, when the matureprotein is not naturally lipidated.

The mature protein encoded by the second nucleic acid sequence generallyis a lipoprotein, preferably an antigenic lipoprotein, and morepreferably is the mature OspC lipoprotein of a Borrelia species,preferably a strain of B. burgdorferi, more preferably a strain of B.burgdorferi selected from the OspC sub-type families. In anotherpreferred embodiment, the mature protein is the mature PspA protein, oran immunogenic fragment thereof, of a strain of S. pneumoniae. In yetanother preferred embodiment, the mature protein is UreA or UreB proteinof a strain of H. pylori. Similarly, the signal sequence of the OspAprotein of a Borrelia strain encoded by the first nucleic acid sequencepreferably is that of a strain of B. burgdorferi, more preferably astrain of B. burgdorferi selected from the B31, ACA1 and Ip90 familiesof strains.

The hybrid gene provided herein may be assembled into an expressionvector, preferably under the control of a suitable promoter forexpression of the mature lipoprotein, in accordance with a furtheraspect of the invention, which, in a suitable host organism, such as E.coli, causes initial translation of a chimeric molecule comprising theleader sequence and the desired heterologous protein in lipidated form,followed by cleavage of the chimeric molecule by signal peptidase II andattachment of lipid moieties to the new terminus of the protein, wherebythe mature lipoprotein is expressed in the host organism.

The present invention provides, for the first time, a hybrid nucleicacid molecule which permits the production of recombinant lipidatedprotein, e.g., recombinant lipidated OspC of a Borrelia species,recombinant lipidated PspA of a strain of S. pneumoniae or recombinantlipidated UreA or UreB of a strain of H. pylori, to be obtained.Accordingly, in a further aspect of this invention, there is provided ahybrid nucleic acid molecule, comprising a first nucleic acid sequenceencoding a lipoprotein, preferably an OspC lipoprotein of a Borreliaspecies, more preferably a strain of B. burgdorferi, still morepreferably a strain of B. burgdorferi selected from the OspC sub-typefamilies; or encoding a PspA lipoprotein of a strain of S. pneumoniae orimmunogenic fragment thereof; or encoding a UreA or UreB lipoprotein ofa strain of H. pylori; and a second nucleic acid sequence encoding asignal sequence of an expressed protein heterologous to the proteinencoded by the first nucleic acid sequence and coupled in translationalopen reading frame relationship with said first nucleic acid sequence,preferably encoding the signal sequence of an OspA protein of a Borreliaspecies.

As described above, the hybrid gene can be assembled into an expressionvector under the control of a suitable promoter for expression of thelipoprotein, which, in a suitable host organism, such as E. coli, causesexpression of the lipoprotein from the host organism.

It has also surprisingly been found that enhanced immunogenicity can beobtained by a recombinant lipoprotein when the lipoprotein is expressedby a hybrid or chimeric gene comprising a first nucleic acid sequenceencoding a leader or signal sequence and a second nucleic acid sequenceencoding the protein portion of the lipoprotein, wherein the first andsecond sequences do not naturally occur together.

Accordingly, the present invention also provides a recombinantlipoprotein expressed by a hybrid or chimeric gene comprising a firstnucleic acid sequence encoding a leader or signal sequence contiguouswith a second nucleic acid sequence encoding a protein portion of thelipoprotein, and the first and second sequences do not naturally occurtogether. The first and second sequences are preferably coupled in atranslational open reading frame relationship. The first sequence canencode a leader sequence of a Borrelia lipoprotein, preferably theleader sequence of ospA; and the second sequence can encode a proteincomprising an antigen, preferably OspC, PspA, UreA, UreB or animmunogenic fragment thereof. The first and second sequences can bepresent in a gene; and the gene and/or the first and second sequencescan be in a suitable vector for expression.

The vector can be a nucleic acid in the form of, e.g., plasmids,bacteriophages and integrated DNA, in a bacteria, most preferably oneused for expression, e.g. E. coli, Bacillus subtilis, Salmonella,Staphylocoocus, Streptococcus, etc., or one used as a live vector, e.g.Lactobacillus, Mycobacterium, Salmonella, Streptococcus, etc. When anexpression host is used the recombinant lipoprotein can be obtained byharvesting product expressed in vitro; e.g., by isolating therecombinant lipoprotein from a bacterial extract. The gene canpreferably be under the control of and therefore operably linked to asuitable promoter; and the promoter can either be endogenous to thevector, or be inserted into the vector with the gene.

The invention further provides vectors containing the nucleic acidencoding the recombinant lipoprotein and methods for obtaining therecombinant lipoproteins and methods for preparing the vector.

As mentioned, the recombinant lipoprotein can have enhancedimmunogenicity. Thus, additional embodiments of the invention provideimmunogenic or vaccine compositions for inducing an immunologicalresponse, comprising the isolated recombinant lipoprotein, or a suitablevector for in vivo expression thereof, or both, and a suitable carrier,as well as to methods for eliciting an immunological or protectiveresponse comprising administering to a host the isolated recombinantlipoprotein, the vector expressing the recombinant lipoprotein, or acomposition containing the recombinant lipoprotein or vector, in anamount sufficient to elicit the response.

Documents cited in this disclosure, including the above-referencedapplications, provide typical additional ingredients for suchcompositions, such that undue experimentation is not required by theskilled artisan to formulate a composition from this disclosure. Suchcompositions should preferably contain a quantity of the recombinantlipoprotein or vector expressing such sufficient to elicit a suitableresponse. Such a quantity of recombinant lipoproprotein or vector can bebased upon known amounts of antigens administered. For instance, ifthere is a known amount for administration of an antigen correspondingto the second sequence expressed for the inventive recombinantlipoprotein, the quantity of recombinant lipoprotein can be scaled toabout that known amount, and the amount of vector can be such as toproduce a sufficient number of colony forming units (cfu) so as toresult in in vivo expression of the recombinant lipoprotein in aboutthat known amount. Likewise, the quantity of recombinant lipoprotein canbe based upon amounts of antigen administered to animals in the examplesbelow and in the documents cited herein, without undue experimentation.

The present invention also includes, in other aspects, processes for theproduction of a recombinant lipoprotein, by assembly of an expressionvector, expression of the lipoprotein from a host organism containingthe expression vector, and optionally isolating and/or purifying theexpressed lipoprotein. The isolating/purifying can be so as to obtainrecombinant lipoprotein free from impurities such as lipopolysaccharidesand other bacterial proteins.

The present invention further includes immunogenic compositions, such asvaccines, containing the recombinant lipoprotein as well as methods forinducing an immunological response.

BRIEF DESCRIPTION OF DRAWING

In the following detailed description, reference is made to theaccompanying drawings, wherein:

FIG. 1 is a schematic representation of a procedure for assemblingplasmid pLF100;

FIG. 2 is a schematic representation of a procedure for assemblingplasmid vectors pPko9a (strain Pko) and pB319a (strain B31);

FIG. 3 is a schematic representation of the procedure employed for theisolation and purification of lipidated OspC;

FIG. 4 is a schematic representation of the procedure employed for theisolation and purification of non-lipidated OspC for comparativepurposes;

FIG. 5 shows an SDS-PAGE analysis of lipidated OspC produced herein atvarious stages of the purification procedure illustrated schematicallyin FIG. 3;

FIG. 6 shows an SDS-PAGE analysis of non-lipidated OspC produced hereinat various stages of the purification procedure as described in WO91/09870;

FIG. 7 is a graphical representation of the immune response of miceimmunized with OspC formulations containing antigen from two OspCsub-types as measured in an anti-OspC ELISA assay;

FIG. 8 is a graphical representation of the immune response of miceimmunized with a two sub-type OspC formulation that contains alumadjuvant as measured in an anti-OspC ELISA assay;

FIG. 9 is a schematic representation of a procedure for assemblingplasmid vector pPA321-L;

FIG. 10 is a schematic representation of a procedure for assemblingplasmid vector pPA321-NL;

FIG. 11 is a schematic representation of the procedure employed for theisolation and purification of lipidated PspA;

FIG. 12 is a schematic representation of the procedure employed for theisolation and purification of non-lipidated PspA for comparativepurposes;

FIG. 13 shows an SDS-PAGE analysis of lipidated PspA produced herein atvarious states of the expression and host cell fractionation procedureillustrated schematically in FIG. 11;

FIG. 14 shows an SDS-PAGE analysis of non-lipidated PspA produced hereinat various stages of the expression and host cell fractionationprocedure illustrated schematically in FIG. 12; and

FIG. 15 shows an SDS-PAGE analysis of the PspA column chromatographyresults illustrated schematically in FIGS. 11 and 12.

DETAILED DESCRIPTION OF INVENTION

As noted above, the present invention is concerned with the use of anucleic acid sequence encoding the OspA signal sequence to expresslipidated proteins heterologous to OspA protein, preferably an OspCprotein of a Borrelia species, a PspA protein or portion thereof of astrain of S. pneumoniae, or a UreA or UreB protein of a strain of H.pylori, and to the use of a nucleic acid sequence encoding the signalsequence of a protein heterologous to the protein to be expressed, toexpress the lipidated OspC protein of a Borrelia species or thelipidated PspA protein of a strain of S. pneumoniae.

The leader amino acid sequence and encoding DNA sequence for the ACAstrain of B. burgdorferi are as follows:M   K   K   Y   L   L   G   I   G (SEQ ID NO: 1)L   I   L   A   L   I   A   C ATG AAA AAA TAT TTA TTG GGA ATA GGT (SEQID NO: 2) CTA ATA TTA GCC TTA ATA GCA TGCThe corresponding leader amino acid sequences and encoding DNA sequencesfor the ospA of other strains of B. burgdorferi are known in the art andmay be employed in the present invention from this disclosure, withoutany undue experimentation.

A hybrid gene molecule is assembled comprising the OspA leader encodingsequence and the gene encoding the heterologous protein to be expressed,preferably the ospc or pspA gene, arranged in translationalreading-frame relationship with the ospA gene fragment.

For production of the lipidated protein, the appropriate hybrid genemolecule can be incorporated into a suitable expression vector and theresulting plasmid incorporated into an expression strain of E. coli orother suitable host organism. The vector can also be a bacteriophage orintegrated DNA.

The lipidated protein is expressed by the cells during growth of thehost organism. The lipidated protein may be recovered from the hostorganism in purified form by any convenient procedure which separatesthe lipidated protein in undenatured form. One schematic of a procedurein accordance with this aspect of the invention is shown in FIG. 3.

Following cell growth and induction of protein, the cells are subjectedto freeze-thaw lysis and DNase I treatment. The lysate is treated with adetergent which is selective for solubilization of the recombinantlipidated protein, in preference to the other bacterial proteins in thelysate. While the present invention preferably utilizes polyethyleneglycol tert-octylphenyl ether having the formulat-Oct-C₆H₄-(OCH₂CH₂)_(x)OH wherein x=7-8 as the detergent (commerciallyavailable as, and hereinafter referred to as, TRITON™ X-114), othermaterials may be used exhibiting a similar selective solubility for thelipidation protein as well as the phase separation property under mildconditions, as discussed below.

Following addition of the TRITON™ X-114, the mixture is warmed to a mildtemperature elevation of preferably about 35° C. to 40° C., at whichtime the solution becomes cloudy as phase separation occurs. Thepurification procedure for such phase separation should occur underconditions to avoid any substantial denaturing or any other substantialimpairment of the immunological properties of the recombinantlipoprotein.

Centrifugation of the cloudy mixture results in separation of themixture into three phases, namely a detergent phase containing about 50%or more of the recombinant lipidate protein and a small amount(approximately 5 wt %) of other proteins, an aqueous phase containingthe balance of the other proteins, and a solid pellet of cell residue.The detergent phase is separated from the aqueous phase and the solidpellet for further processing.

Final purification of the protein preferably is effected by processingof the detergent phase to provide a recombinant lipidated protein havinga purity of at least about 80 wt %, and which is substantially free fromother contaminants such as bacterial proteins, and lipopolysaccharides(LPS), and which has endotoxin levels compatible with humanadministration.

Such purification is conveniently effected by column chromatography.Such chromatographic purification may include a first chromatographicpurification using a first chromatographic column having the pH, ionicstrength and hydrophobicity to bind bacterial proteins, but not therecombinant lipidated protein.

Such first chromatographic purification may be effected by loading thedetergent phase onto the first chromatographic column and theflow-through, which contains the purified lipidated protein, iscollected. The bound fraction contains substantially all the bacterialprotein impurities from the detergent phase. The chromatography mediumused for such first purification operation may be a DEAE-Sephacel orDEAE-Sepharose column.

The flow-through from the first chromatographic purification operationmay be subjected to further purification on a second chromatographiccolumn. The flow-through is loaded onto the column having the pH, ionicstrength and hydrophobicity which will selectively bind the recombinantlipidated protein to the second chromatographic column, while bacterialcontaminants and LPS pass through the column. The chromatography mediumfor the second chromatographic column may be S-Sepharose.

Preferably the recombinant lipoprotein is purified to 80% purity or togreater than 80% purity, e.g., 85-90% or even 90-95% or greater than 95%purity. The lipidated proteihaceous material can then be formulated intoimmunogenic compositions, preferably vaccines.

The vaccine or immunogenic composition elicits an immune response in ahost subject which produces an immunological response, such asantibodies which may be opsonizing or bactericidal. Should a subjectimmunized with a recombinant lipoprotein of the invention then bechallenged, such immunological response can inactivate the challengeorganism. Furthermore, opsonizing or bactericidal antibodies may alsoprovide protection by alternative mechanisms.

Immunogenic compositions including vaccines can be prepared asinjectables, as liquid solutions or emulsions, or as formulating fororal, nasal or other orifice administration e.g., vaginal, rectal, etc.Oral formulations can be liquid solutions, emulsions and the like, e.g.,elixers, or solid preparations, e.g., tablets, caplets, capsules, pills,liquid-filled-capsules, gelatin and the like. Nasal preparations can beliquid and can be administered via aerosol, squeeze spray or pump spraydispensers. Documents cited herein provide exemplary formulation typesand ingredients therefor, including the applications cited above.

The immunogens can be mixed with pharmaceutically acceptable excipientswhich are compatible with the immunogens. Such excipients may includewater, saline, dextrose, glycerol, ethanol, and combinations thereof.The immunogenic compositions and vaccines may further contain auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,or adjuvants to enhance the effectiveness thereof. Immunogeniccompositions and vaccines may be administered parenterally, by injectionsubcutaneously or intramuscularly. The immunogenic preparations andvaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective,immunogenic or protective. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theimmune system of the individual to synthesize antibodies, and, ifneeded, to produce a cell-mediated immune response. Precise amounts ofactive ingredient required to be administered depend on the judgment ofthe practitioner, taking into account such factors as the age, weight,sex, condition of the host or patient to whom there is to beadministration. However, suitable dosage ranges are readily determinableby one skilled in the art and may be of the order of micrograms of theimmunogens. Suitable regimes for initial administration and boosterdoses are also variable, but may include an initial administrationfollowed by subsequent administrations. The dosage may also depend onthe route of administration and will vary according to the size of thehost.

The concentration of the immunogens in an immunogenic compositionaccording to the invention is in general about 1 to about 95%. A vaccineor immunogenic composition which contains antigenic material of only onepathogen is a monovalent vaccine. Vaccines or immunogenic compositionswhich are multivalent or which contain antigenic material of severalpathogens (also known as combined vaccines or combined imunogeniccompositions) also belong to the present invention. Such combinedvaccines or immunogenic compositions contain, for example, material fromvarious pathogens or from various strains of the same pathogen, or fromcombinations of various pathogens.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines orimmunogenic compositions. Intrinsic adjuvants, such aslipopolysaccharides, normally are the components of the killed orattenuated bacteria used as vaccines or immunogenic compositions.Extrinsic adjuvants are immunomodulators which are typicallynon-covalently linked to antigens and are formulated to enhance the hostimmune responses. Some of these adjuvants are toxic, however, and cancause undesirable side-effects, making them unsuitable for use in humansand many animals. Indeed, only alum is routinely used as an adjuvant inhuman and veterinary vaccines.

In view of the difficulties associated with the use of adjuvants, it isthus an advantage of the present invention that the recombinantlipidated proteins are the most immunogenic forms, and are capable ofeliciting immune responses both without any adjuvant and with alum.

The following examples illustrate but do not limit the scope of theinvention disclosed in this specification.

EXAMPLES Example 1

Construction of a Vector Containing a Gene Encoding the OspA LeaderSequence

Plasmid pBluescript KS+ (Stratagene) was digested with XbaI and BamHIand ligated with a 900 bp XbaI-BamHI DNA fragment containing thecomplete coding region of B. burgdorferi strain ACA1 ospA gene, to forma lipoprotein fusion vector pLF100. This procedure is shownschematically in FIG. 1.

The vector pLF100 has been deposited with the American Type CultureCollection at Rockville, Md. on Feb. 2, 1995 under Accession No. 69750.This deposit was made under the terms of the Budapest Treaty.

Example 2

construction of a pET9a Expression Vector Containing a Hybrid ospA-ospCGene

Specifically designed oligonucleotide primers were used in a polymerasechain reaction (PCR) to amplify the portion of the ospc gene downstreamfrom the cysteine-encoding codon terminating the signal-peptiderecognition-encoding sequence to the C-terminal end of the coding regionfrom the Pko and B31 strains of B. burgdorferi.

The 5′-end primer had the nucleotide sequences respectively for the Pkoand B31 strains: 5′-GGC GCG CAT GCA ATA ATT (Pko) (SEQ ID NO: 3) CAG GGAAAG G-3′ 5′-GGC GCG CAT GCA ATA ATT (B31) (SEQ ID NO: 4) CAG GGA AAGA-3′ while the 3′-end primer had the nucleotide sequence: 5′-CGC GGA TCCTTA AGG TTT (B31 & (SEQ ID NO: 5) TTT TGG-3′ Pko)

The PCR amplification was effected in a DNA Thermal Cycler(Perkins-Elmer Cetus) for 25 cycles with denaturation for 30 secs at 94°C., annealing at 37° C. for 1 minute and extension at 72° C. for 1minute. A final extension was effected at 72° C. for 5 minutes at thecompletion of the cycles. The product was purified using a Gene Clean IIkit (B10 101) and the purified material was digested with SphI andBamHI. This procedure introduced a silent mutation in the Pko ospC genewhich changes the codon for amino acid 60 of the mature protein from ATTto ATA.

The materials produced for the Pko and B31 B. burgdorferi strains werehandled identically from this point on and hence only the furtherhandling of the Pko strain OspC material is described.

The plasmid pLF100 (Example 1) was digested with SphI and BamHI and theamplified PKo sequence was ligated into the plasmid to form plasmid ppko100 (pB31 100 for the B31 strain) containing a hybrid ospA/ospC gene.The hybrid gene was excised from plasmid pPko 100 by digestion with NdeIand BamHI and cloned into the NdeI and BamHI sites of the plasmid vectorpET9 to place the ospA/ospC hybrid gene under control of a T7 promoterand efficient translation initiation signals from bacteriophage T7, asseen in FIG. 2. The resulting plasmid is designated pPko9a (pB319a forthe B31 strain).

Example 3

Expression and Purification of Lipidated OspC.

Plasmid pPko9a, prepared as described in Example 2, was used totransform E. coli strains BL21(DE3) (pLysS) and HMS174(DE3)(pLysS). Thetransformed E. coli was inoculated into LB media with 30 μg/ml kanamycinsulfate and 25 μg/ml of chloramphenicol at a rate of 12 ml of culturefor every liter prepped. The culture was grown overnight in a flaskshaker at 37° C.

The next morning, 10 ml of overnight culture medium was transferred to 1L of LB media containing 30 μg/ml of kanamycin sulfate and the culturewas grown in a flask shaker at about 37° C. to a level of OD₆₀₀=0.6-1.0(although growth up to OD₆₀₀=1.5 can be effected), in approximately 3-5hours.

To the culture medium was added isopropylthiogalactoside (IPTG) to afinal concentration of 0.5 mM and the culture medium was grown for afurther two hours at about 30° C. The cultures were harvested andsamples analyzed on Coomassie stained SDS-PAGE gels (FIG. 5). Theculture medium was cooled to about 4° C. and centrifuged at 10,000×G for10 minutes. The supernatant was discarded while the cell pellet wascollected. Purified lipidated OspC was recovered from the pellet byeffecting the procedure shown schematically in FIG. 3 and describedbelow.

The cell pellet first was resuspended in 1/10 the volume of PBS. Thecell suspension was frozen and stored at −20° C. or below, if desired.Following freezing of the cell suspension, the cells were thawed to roomtemperature (about 20° C. to 25° C.) which causes the cells to lyse.DNase I was added to the thawed material to a concentration of 1 μg/mland the mixture was incubated for 30 minutes at room temperature, whichresulted in a decrease in the viscosity of the material.

The incubated material was chilled on ice to a temperature below 10° C.and Triton™ X-114 was added as a 10 wt % stock solution, to a finalconcentration of 0.3 to 1 wt %. The mixture was kept on ice for 20minutes. The chilled mixture next was heated to about 37° C. and held atthat temperature for 10 minutes.

The solution turned very cloudy as phase separation occurred. The cloudymixture then was centrifuged at about 20° C. for 10 minutes at 12,000×G,which caused separation of the mixture into a lower detergent phase, anupper clear aqueous phase and a solid pellet. Analysis of the phasesfractionated by SDS-PAGE (FIG. 5) revealed that the OspC partitionedinto the detergent phase, showing that it is in lipidated form. Thedetergent phase was separated from the other two phases and cooled to 4°C., without disturbing the pellet.

Buffer A, namely 50 mM Tris pH 7.5, 2 mM EDTA and mM NaCl and 0.3%polyethylene glycol tert-octylphenyl ether having the formulat-Oct-C₆H₄—(OCH₂CH₂)_(x)OH wherein x=9-10 as the detergent (commerciallyavailable as, and hereinafter referred to as, Triton™ X-100), was addedto the cooled detergent phase to reconstitute back to ⅓ the originalvolume. The resulting solution may be frozen and stored for laterprocessing as described below or may be immediately subjected to suchprocessing.

A DEAE-Sepharose CL-6B column was prepared in a volume of 1 ml/10 ml ofdetergent phase and was washed with 2 volumes of Buffer C, namely 50 mMTris pH 7.5, 2 mM EDTA, 1 M NaCl, 0.3 wt % Triton™ X-100, and then with4 volumes of Buffer B, namely 50 mM Tris pH 7.5, 2 mM EDTA, 0.3 wt %Triton™ X-100.

The detergent phase then was loaded onto the column and the flow-throughcontaining the OspC, was collected. The column was washed with 2 volumesof Buffer B and the flow-through again was collected. The combinedflow-through was an aqueous solution of purified OspC, which may befrozen for storage.

The column may be freed from bacterial proteins for reuse by elutingwith 4 volumes of Buffer C.

Further and final purification of the flow-through from theDEAE-Sepharose column was effected by chromatography on S-Sepharose FastFlow. The flow-through from the DEAE-Sepharose column first wasacidified to pH 4.3 by the addition of 1 M citric acid and the acidifiedmaterial was loaded onto the S-Sepharose column. The S-Sepharose FastFlow column had been washed with 3 column volumes of Buffer A and thenwith 5 column volumes of Buffer A made up to pH 4.3. The OspC binds tothe column. The loaded column was washed with 4 column volumes of pH 4.3Buffer A followed by 4 column volumes of pH 5.5 Buffer A.

Highly-purified OspC was eluted from the column using Buffer A, adjustedto pH 6.0 with 1N HCl. A schematic of the purification process describedin this Example is shown in FIG. 3.

The aqueous solution of highly purified lipidated ospC obtained by thechromatography procedures was analyzed by Coomassie stained gels (FIG.5), and confirmed to be OspC in highly purified form by immunoblotanalysis using rabbit anti-OspC polyclonal antiserum. The purity of theproduct was estimated to be greater than 80%.

By this procedure, about 2 to 4 mg of pure OspC was recovered from a 1liter culture of the BL21 host and about 1 to 2 mg of pure OspC wasrecovered from a 1 liter culture of the HMS 174 host.

Example 4

Expression and Purification of Non-Lipidated OspC.

E. coli JM 109 transformants containing plasmid vector containingchromosomal gene fragment encoding non-lipidated OspC were prepared andgrown as described in WO 91/09870. The cultures were harvested, theculture medium centrifuged at 10,000×G for 10 minutes at 4° C., thesupernatant discarded and the pellet collected.

The cell pellet was first resuspended in lysis buffer A, namely 50 nMTris-HCI pH 8.0, 2 mM EDTA, 0.1 mM DTT, 5% glycerol and 0.4 mg/mllysozyme, and the suspension stirred for 20 minutes at room temperature.TRITON™ X-100 then was added to the cell suspension to a concentrationof 1 wt %, DNase I was added to a concentration of 1 μg/ml, and thesuspension stirred at room temperature for a further 20 minutes toeffect cell lysis. Sodium chloride next was added to the cell suspensionto a concentration of 1M and the suspension again stirred at 4° C. for afurther 20 minutes. The suspension then was centrifuged at 20,000×G for30 minutes, the resultant supernatant separated from the pellet and thepellet was discarded.

The separated supernatant was dialyzed against a buffer comprising 50 mMTris pH 8, 2 mM EDTA. The supernatant next was loaded onto aDEAE-Sepharose CL-6B column and the non-lipidated OspC was collected inthe column flow-through. The flow-through was dialyzed against a 0.1 Mphosphate buffer, pH 6.0.

The dialyzed flow-through next was bound to a S-Sepharose fast flowcolumn equilibrated with 0.1M phosphate buffer, pH 6.0. Purifiednon-lipidated OspC then was eluted from the S-Sepharose column using thedialysis buffer with 0.15 M NaCl added. A schematic of the purificationprocess is shown in FIG. 4.

The aqueous solution of highly purified non-lipidated OspC was analyzedby Coomassie stained gels (FIG. 6). The purity of the product wasestimated to be greater than 80%.

Example 5

Immunogenicity of Recombinant Lipidated OspC.

Purified recombinant lipidated OspC, prepared as described in Example 3,was tested for immunogenicity in mice and compared to that fromnon-lipidated OspC prepared as described in Example 4. For this study, 4to 8 week old female C3H/He mice were immunized on day 0 and boosted onday 21 and 42. All animals were given 1 μg each of OspC expressed fromthe B31 and Pko genes per dose. Both lipidated and non-lipidated formsof the antigen were tested. Formulations were tested with and withoutalum as an adjuvant.

Representative animals were exsanguinated on days 21, 42, 63 and 91.ELISA testing was performed on these sera using purified non-lipidatedOspC as the coating antigen.

The test results from mice immunized with unadjuvanted antigen (FIG. 7)show that only animals immunized with the lipidated antigen make adetectable ELISA response. However, the immune response of animalsimmunized antigens formulated on alum (FIG. 8) shows that two types ofantigen give comparible ELISA responses and these responses develop morerapidly.

Example 6

Construction of a pET9a Expression Vector Containing a Hybrid ospA/pspAGene

Specifically designed oligonucleotide primers were used in a PCRreaction to amplify the portion of the pspA gene of interest (in thiscase from amino acid 1 to 321) from the S. pneumoniae strain R×1.

The 5′-end primer had the nucleotide sequence: 5′-GGG ACA GCA TGC GAAGAA (PspN1). (SEQ ID NO: 6) TCT CCC GTA GCC AGT-3′

The 3′-end primer had the nucleotide sequence: 5′-GAT GGA TCC TTT TGG(PspC370). (SEQ ID NO: 7) TGC AGG AGC TGG TTT-3′

The PCR reaction was as follows: 94° C. for 30 seconds to denature DNA;42° C. for one minute for annealing DNA; and 72° C. for one minute forextension of DNA. This was carried out for 25 cycles, followed by a 5minute extension at 72° C. This procedure introduced a stop codon atamino acid 315. The PCR product was purified using the Gene Clean IImethod (Bio101), and digested with SphI and BamHI.

The plasmid pLF100 (Example 1) was digested with SphI and BamHI and theamplified pspA gene was ligated to this plasmid to form the plasmidpLF321, which contained the hybrid ospA-pspA gene. The hybrid gene wasexcised from pLF321 by digestion with NdeI and BamHI and cloned into theNdeI and BamHI sites of the plasmid vector pET9a to place the ospA-pspAhybrid gene under the control of a T7 promoter. The resulting plasmid iscalled pPA321-L. This process is shown schematically in FIG. 9.

Example 7

Construction of a pET9a Expression vector Containing the pspA Gene

Specifically designed oligonucleotide primers were used in a PCRreaction to amplify the portion of the pspA gene of interest (in thiscase from amino acid 1 to 321) from the S. pneumoniae strain R×1 usingplasmid pPA321-L of Example 6.

The 5′-end primer had the nucleotide sequence: 5′-GCT CCT GCA TAT GGAAGA (PspNL-2) (SEQ ID NO: 8) ATC TCC CGT AGC C-3′

The 3′-end primer had the nucleotide sequence: 5′-GAT GGA TCC TTT TGG(PspC370). (SEQ ID NO: 7) TGC AGG AGC TGG TTT-3′

The PCR reaction was as follows: 94° C. for 30 seconds to denature DNA;and 72° C. for one minute for annealing and extension of DNA. This wascarried out for 25 cycles, which was followed by a 5 minute extension at72° C. This procedure introduced a stop codon at amino acid 315. The PCRproduct was purified using the Gene Clean II method (Bio 101), anddigested with NdeI and BamHI. The digested PCR product was cloned intothe NdeI and BamHI sites of the plasmid vector pET9a to place the pspAgene under the control of a T7 promoter. The resulting plasmid is calledpPA321-NL. This process is shown scematically in FIG. 10.

Example 8

Expression and Purification of Lipidated PspA

Plasmid pPA321-L was used to transform E. coli strain BL21(DE3)pLyS. Thetransformed E. coli was inoculated into LB media containing 30 μg/mlkanamycin sulfate and 25 μg/ml chloramphenicol. The culture was grownovernight in a flask shaker at 37° C.

The following morning 50 ml of overnight culture was transferred to 1LLB media containing 30 μg/ml kanamycin sulfate and the culture was grownin a flask shaker at 37° C. to a level of OD 600 nm of 0.6-1.0, inapproximately 3-5 hours. To the culture medium was added IPTG to a finalconcentration of 0.5 mM and the culture was grown for an additional twohours at 30° C. The cultures were harvested by centrifugation at 4° C.at 10,000×G and the cell pellet collected. Lipidated PspA was recoveredfrom the cell pellet.

The cell pellet was resuspended in PBS at 30 g wet cell paste per literPBS. The cell suspension was frozen and stored at −20° C. The cells werethawed to room temperature to effect lysis. DNaseI was added to thethawed material at a final concentration of 1 μg/ml and the mixtureincubated for 30 minutes at room temperature, which resulted in adecrease in viscosity of the material.

The material was then chilled in an ice bath to below 10° C. and Triton™X-114 was added as a 10% stock solution to a final concentration of 0.3to 1%. The mixture was kept on ice for 20 minutes. The chilled mixturewas then heated to 37° C. and held at that temperature for 10 minutes.This caused the solution to become very cloudy as phase separationoccurred. The mixture was then centrifuged at about 20° C. for 10minutes at 12,000×G, which caused a separation of the mixture into alower detergent phase, an upper clear aqueous phase and a pellet. Thelipidated PspA partitioned into the detergent phase. The detergent phasewas separated from the other two phases, diluted 1:10 with a buffercomprising 50 mM Tris, 2 mM EDTA, 10 mM NaCl pH 7.5, and was stored at−20° C.

A Q-Sepharose column was prepared in a volume of 1 ml per 5 ml diluteddetergent phase. The column was washed with 2 column volumes of a buffercomprising 50 mM Tris, 2 mM EDTA, 0.3% Triton™ X-100, 1M NaCl pH 4.0,and then equilibrated with 5 to 10 column volumes 50 mM Tris, 2 mM EDTA,0.3% Triton™ X-100, 10 mM NaCl pH 4.0. The pH of the diluted detergentphase material was adjusted to 4.0, at which time a precipitationoccurred. This material was passed through a 0.2 μM cellulose acetatefiltering unit to remove the precipitated material. The filtered diluteddetergent phase was applied to the Q-Sepharose column and the flowthrough (containing PA321-L) was collected. SDS-PAGE analysis of thisstep is shown in FIG. 15. The column was washed with 1-2 column volumesof 50 mM Tris, 2 mM EDTA, 0.3% Triton™ X-100, 10 mM NaCl pH 4.0, and theflow through was pooled with the previous flow through fraction. The pHof the flow through pool was adjusted to 7.5. The bound material,contaminating E. coli proteins, was eluted from the Q-Sepharose with 2column volumes of 50 mM Tris, 2 mM EDTA, 0.3% Triton™ X-100, 1M NaCl pH4.0. A schematic of the purification process described in this Exampleis shown in FIG. 11.

Example 9

Expression and Purfication of Non-Lipidated PspA

Plasmid pPA321-NL was used to transform E. coli strain BL21(DE3)pLyS.The transformed E. coli was incolulated into LB media containing 30μg/ml kanamycin sulfate and 25 μg/ml chloramphenicol. The culture wasgrown overnight in a flask shaker at 37° C.

The following morning 50 ml of overnight culture was transferred to 1LLB media containing 30 μg/ml kanamycin sulfate and the culture was grownin a flask shaker at 37° C. to a level of OD 600 nm of 0.6-1.0, inapproximately 3-5 hours. To the culture medium was added IPTG to a finalconcentration of 0.5 mM and the culture was grown for an additional twohours at 30° C. The cultures were harvested by centrifugaton at 4° C. at10,000×G and the cell pellet collected. Non-lipidated PspA was recoveredfrom the cell pellet.

The cell pellet was resuspended in PBS at 30 g wet cell paste per literPBS. The cell suspension was frozen and stored at −20° C. The cells werethawed to room temperature to effect lysis. DNaseI was added to thethawed material at a final concentration of 1 μg/ml and the mixtureincubated for 30 minutes at room temperature, which resulted in adecrease in viscosity of the material. The mixture was centrifuged at 4°C. at 10,000×G, and the cell supernatant saved, which containednon-lipidated PspA. The pellet was washed with PBS, centrifuged at 4° C.at 10,000×G and the cell supernatant pooled with the previous cellsupernatant.

A MonoQ column (Pharmacia) was prepared in a volume of 1 ml per 2 mlcell supernatant. The column was washed with 2 column volumes of abuffer comprising 50 mM Tris, 2 mM EDTA, 1M NaCl pH 7.5, and thenequilibrated with 5 to 10 column volumes of a buffer comprising 50 mMTris, 2 mM EDTA, 10 mM NaCl pH 7.5. The cell supernatant pool wasapplied to the Q-Sepharose column and the flow through was collected.The column was washed with 2-5 column volumes of 50 mM Tris, 2 mM EDTA,10 mM NaCl pH 7.5, and the flow through pooled with the previousflowthrough.

The elution of bound proteins began with the first step of a 5-10 columnvolume wash with 50 mM Tris, 2 mM EDTA, 100 mm NaCl pH 7.5. The secondelution step was a 5-10 column volume wash with 50 mM Tris, 2 mM EDTA,200 mM NaCl pH 7.5. The non-lipidated PspA was contained in thisfraction. SDS-PAGE analysis of this step is shown in FIG. 15. Theremaining bound contaminating proteins were removed with 50 mM Tris and2 mM EDTA pH 7.5 with 300 mM-1M NaCl.

A schematic of the purification process described in this Example isshown in FIG. 12.

Example 10

Immunogenicity of Recombinant Lipidated PspA

Purified recombinant lipidated PspA, prepared as described in Example 8,was tested for immunogenicity in mice and compared to that fromnon-lipidated PspA prepared as described in Example 9. For this study,CBA/N mice were immunized subcutaneously in the back of the neck with0.5 ml of the following formulations at the indicated PspA antigenconcentrations. PspA Antigen Formulation Concentration Native PspAmolecule of the RX1 200 ng/ml strain (Native RX1) Non-LipidatedRecombinant PspA 200 and 1000 ng/ml (pPA-321-NL) Alone in PBS*Non-Lipidated Recombinant PspA 200 and 1000 ng/ml (pPA-321-NL) Adsorbedto Alum Lipidated Recombinant PspA (pPA- 200 and 1000 ng/ml 321-L) Alonein PBS Lipidated Recombinant PspA 200 and 1000 ng/ml (pPA0321-NL)Adsorbed to Alum* Alum* 0 ng/ml PBS 0 ng/ml*Alum was Hydrogel at a concentration of 200 μg/ml

Four mice were immunized on days 0 and 21 for each dosage of theformulations. The mice were then bled on day and subsequently challengedwith S. pneumoniae of A66 strain. The days of survival after challengefor the mice were recorded and surviving mice were bled on days 36, 37,42 and 46. From these subsequent bleeds the blood was assayed for thenumber of colony forming units (CFU) of S. pneumoniae/ml. The sera takenon day 35 were assayed by ELISA for antibodies against PspA using ELISA.The days to death for the challenged mice are shown in the followingtable. Survival in Immune and Non-Immune CBA/N Mice ImmunizationEfficacy dose Days to P value time Alive: P value Group Antigen in μgAlum Death to death* Dead Survival* #1A pPA-321-L 1.0 − 4x > 14 0.01 4:00.01 #1B PpA-321-L 0.2 − 4x > 14 0.01 4:0 0.01 #2A pPA-321-L 1.0 + 4x >14 0.01 4:0 0.01 #2B pPA-321-L 0.2 + 4x > 14 0.01 4:0 0.01 #3ApPA-321-NL 1.0 − 1, 1, 2, 2 n.s. 0:4 n.s. #3B pPA-321-NL 0.2 − 1, 1, 2,≧15 n.s. 1:3 n.s. #4A pPA-321-NL 1.0 + 4x > 14 0.01 4:0 0.01 #4BpPA-321-NL 0.2 + 4x > 14 0.01 4:0 0.01 #5 FL-Rx1 0.2 − 4x > 14 0.01 4:00.01 #6 none 0.0 + 1, 1, 3, 6 n.s. 0:4 n.s #7 none 0.0 − 1, 1, 1, ≧15n.s. 1:3 n.s. pooled 0.0 5 × 1, 3, 6, ≧15 — 1:7 noneNote:*indicates versus pooled controls; time to death, by one tailed twosample rank test; survival, by one tailed Fisher Exact test.Calculations have been done using “one tail” since we have neverobserved anti-PspA immunity to consistently cause susceptibility.

The number of CFU in the blood of the mice are shown in the table below.Bacteremia in Immune and Non-Immune CBA/N Mice Immunization Cog₁₀CFUdose in 2 6 7 Group Antigen μg Alum 1 day day day day #1A pPA-321-L 1.0− ≦1.6, 1.9, 2.1, 4x ≦ 1.6 4x ≦ 1.6 n.d. 2.5 #1B pPA-321-L 0.2 − 3x ≦1.6, 1.7 4x ≦ 1.6 4x ≦ 1.6 n.d. #2A pPA-321-L 1.0 + 2x ≦ 1.6, 1.7, 2.93x ≦ 1.6, 1.7 4x ≦ 1.6 n.d. #2B pPA-321-L 0.2 + 2x ≦ 1.6, 1.7, 1.7 4x ≦1.6 4x ≦ 1.6 n.d. #3A pPA-321-NL 1.0 − ≦1.6, 1.7, d, d d, d, d, d d, d,d, d d, d, d, d #3B pPA-321-NL 0.2 − 2x > 7, d, d ≦1.6, d, d, d ≦1.6, d,d, d n.d., d, d, d #4A pPA-321-NL 1.0 + 2x ≦ 1.6, 6.7, >7 3x ≦ 1.6, 1.74x ≦ 1.6 n.d. #4B pPA-321-NL 0.2 + ≦1.6, 1.7, 2.1, 4x ≦ 1.6 4x ≦ 1.6n.d. 2.4 #5 FL-Rx1 0.2 − 2x ≦ 1.6, 2.6, 2.7 4x ≦ 1.6 4x ≦ 1.6 n.d. #6none 0.0 + ≦1.6, 4.1, >7, d ≦1.6, 5.1, d, d 6.1, d, d, d d, d, d, d #7none 0.0 − 1.7, >7, >7, d ≦1.6, d, d, d ≦1.6, d, d, d n.d, d, d, dpooled none 0.0 ≦1.6, 4.1, >7, 2x ≦ 1.6, 5.1, ≦1.6, 6.1, n.d, d, >7, dd, d, d, d, d d, d, d, d, d, d d, d, d, d, dNote:1 colony at the highest concentration of blood calculated out to 47 CFUor Log 1.7. Thus “≦1.6” indicates no colonies counted. >10⁷ indicatesthat the mouse was alive but the number of colonies at the highestdilution was too high to count. “d” indicates the mice had died prior toassay.

These results indicate that the recombinant protein was not protectivewhen injected alone. The recombinant antigen adjuvanted with alum and/orPAM₃Cys lipidation was immunogenic and protective. The native fulllength PspA antigen did not need an adjuvant to be protective. The CFUresults indicate that mice protected by immunization cleared thechallenging S. pneumoniae from the blood in two days.

ELISA analysis of sera taken on day 35 indicated that there was a goodcorrelation between protection of the mice from S. pneumoniae challengeand the induction of measurable antibody responses. No detectableantibody reponses were observed in the sera of mice immunized with thenon-lipidated antigen (pPA-321-NL) in saline or to the negative controlsthat did not contain PspA antigen, (as shown in the table below). Goodantibody responses were detected to the Native R×1 PspA antigen and tothe recombinant PspA when it was lipidated with PAM₃cys and/or adsorbedto alum. ELISA Analysis of Day 35 Mouse Sera PspA Dose PspA Alum (μg/Resulting OD at Indicated Dilution of the Antisera* Antigen Adsorptionmouse 600 1200 2400 4800 9600 19200 pPA-321-L No 0.1 0.885 0.497 0.2710.146 0.075 0.039 (0.082) (0.043) (0.025) (0.017) (0.012) (0.009)pPA-321-L No 0.5 1.857 1.437 1.108 0.750 0.459 0.284 (0.060) (0.137)(0.150) (0.139) (0.092) (0.057) pPA-321-L Yes 0.1 1.373 1.048 0.7450.490 0.288 0.171 (0.325) (0.376) (0.362) (0.304) (0.197) (0.147)pPA-321-L Yes 0.5 1.202 0.787 0.472 0.296 0.162 0.087 (0.162) (0.184)(0.187) (0.102) (0.061) (0.035) pPA-321-NL No 0.1 0.022 0.030 0.0140.007 0.006 0.001 (0.035) (0.060) (0.024) (0.018) (0.005) (0.001)pPA-321-NL No 0.5 0.029 0.014 0.008 0.003 0.002 0.002 (0.035) (0.014)(0.007) (0.004) (0.002) (0.002) pPA-321-NL Yes 0.1 0.822 0.481 0.2780.154 0.082 0.042 (0.181) (0.166) (0.085) (0.051) (0.029) (0.015)pPA-321-NL Yes 0.5 1.017 0.709 0.447 0.253 0.141 0.075 (0.139) (0.128)(0.101) (0.057) (0.034) (0.020) Native RX1 No 0.1 1.367 1.207 0.9220.608 0.375 0.209 (0.084) (0.060) (0.070) (0.077) (0.048) (0.029) NoneNo 0 0.018 0.012 0.009 0.005 0.005 0.005 (0.003) (0.008) (0.003) (0.002)(0.002) (0.002) None Yes 0 0.013 0.009 0.004 0.004 0.001 0.000 (0.006)(0.008) (0.004) (0.003) (0.001) (0.000)*The OD is the mean of the result of the four tested animals and thestandard deviation is in parentheses.

To determine whether protection was at least in part mediated by theanti-PspA antibody responses, a passive experiment was run. BALB/c micewere immunized with 0.5 μg of recombinant lipidated PspA alone orabsorbed to alum, or with recombinant non-lipidated PspA adsorbed toalum on days 0 and 21; and were bled on day 35. The anti-sera werediluted 1:3 or 1:15 in saline and 0.1 ml of the dilution was injectedi.p. into two mice for each dilution. A ⅓ dilution of normal BALB/cmouse serum was used as a negative control. Subsequently one hour afterpassive immunization, the animals were challenged i.v. with the WU2strain of S. pneumoniae (15,000 CFU). Mice passively immunized withanti-PspA sera were protected as compared-to those mice that receiveddilutions of normal mouse sera as shown in the following table. PassiveProtection of BALB/c to WU2 Immunizing Formulation PspA Dose DilutionDays to Death PspA Antigen Alum (μg/animal) of Serum Post ChallengepPA-321-L No 0.5 3   4, >7 15 2, 4 pPA-321-L Yes 0.5 3 >7, >7 15   4, >7pPA-321-NL Yes 0.5 3 2, 4 15 >7, >7 None No 0 3 2, 2

Having thus described in detail certain preferred embodiments of thepresent invention, it is to be understood that the invention defined bythe appended claims is not to be limited by particular details set forthin the above description, as many apparent variations thereof arepossible without departing from the spirit or scope thereof.

1. A hybrid nucleic acid molecule, comprising a first nucleic acidsequence encoding a signal sequence of a lipoprotein and a secondnucleic acid sequence encoding a mature protein, or fragment thereof,which is heterologous to the lipoprotein encoded by said first-nucleicacid sequence, said first nucleic acid sequence being contiguous withsaid second nucleic acid sequence when the mature protein is naturallylipidated, or said first and second nucleic acid sequences beingseparated by one codon coding for one amino acid when the mature proteinis not naturally lipidated.
 2. The hybrid nucleic acid molecule of claim1 wherein said signal sequence is the signal sequence of an OspA proteinof a Borrelia species.
 3. The hybrid nucleic acid molecule of claim 2wherein said first nucleic acid sequence and said second nucleic acidsequence are coupled in a translational open reading frame relationship.4. The hybrid nucleic acid molecule of claim 3 wherein said matureprotein is an OspC lipoprotein of a Borrelia species, or a fragmentthereof.
 5. The hybrid molecule of claim 4 wherein said ospC lipoproteinis that of a strain of B. burgdorferi.
 6. The hybrid molecule of claim 5wherein said strain of B. burgdorferi is selected from the OspC sub-typefamilies.
 7. The hybrid molecule of claim 5 wherein said OspA protein isthat of a strain of B. burgdorferi.
 8. The hybrid molecule of claim 7wherein said strain of B. burgdorferi is selected from the B31, ACA1 andIp90 families of strains.
 9. The hybrid nucleic acid molecule of claim 3wherein said mature protein is a PspA protein of a strain of S.pneumoniae, or a fragment thereof.
 10. The hybrid molecule of claim 9wherein said OspA protein is that of a strain of B. burgdorferi.
 11. Thehybrid molecule of claim 10 wherein said strain of B. burgdorferi isselected from the B31, ACA1 and Ip90 families of strains.
 12. The hybridnucleic acid molecule of claim 3 wherein said mature protein is a UreAprotein of a strain of H. pylori, or a fragment thereof.
 13. The hybridmolecule of claim 12 wherein said ospA protein is that of a strain of B.burgdorferi.
 14. The hybrid molecule of claim 13 wherein said strain ofB. burgdorferi is selected from the B31, ACA1 and Ip90 families ofstrains.
 15. The hybrid nucleic acid molecule of claim 3 wherein saidmature protein is a UreB protein of a strain of H. pylori, or a fragmentthereof.
 16. The hybrid molecule of claim 15 wherein said OspA proteinis that of a strain of B. burgdorferi.
 17. The hybrid molecule of claim16 wherein said strain of B. burgdorferi is selected from the B31, ACA1and Ip90 families of strains.
 18. A hybrid nucleic acid molecule,comprising a first nucleic acid sequence encoding an OspC lipoprotein ofa Borrelia species and a second nucleic acid sequence encoding a signalsequence of an expressed protein heterologous to OspC and coupled intranslational open reading frame relationship with said first nucleicacid sequence.
 19. The hybrid nucleic acid molecule of claim 18 whereinsaid OspC lipoprotein is that of a strain of B. burgdorferi.
 20. Thehybrid nucleic acid molecule of claim 19 wherein said strain of B.burgdorferi is selected from the OspC sub-type families.
 21. A hybridnucleic acid molecule, comprising a first nucleic acid sequence encodinga PspA protein of a strain of S. pneumoniae and a second nucleic acidsequence encoding a signal sequence of an expressed protein heterlogousto PspA and coupled in translational open reading frame relationshipwith said first nucleic acid sequence.
 22. A hybrid nucleic acidmolecule, comprising a first nucleic acid sequence encoding a UreAprotein of a strain of H. pylori and a second nucleic acid sequenceencoding a signal sequence of an expressed protein heterlogous to UreAand coupled in translational open reading frame relationship with saidfirst nucleic acid sequence.
 23. A hybrid nucleic acid molecule,comprising a first nucleic acid sequence encoding a UreB protein of astrain of H. pylori and a second nucleic acid sequence encoding a signalsequence of an expressed protein heterlogous to UreB and coupled intranslational open reading frame relationship with said first nucleicacid sequence.
 24. An expression vector containing the hybrid nucleicacid molecule of claim 1 under control of a promoter for expression ofsaid mature protein.
 25. The expression vector of claim 24 wherein saidmature protein is an OspC lipoprotein of a Borrelia species.
 26. Theexpression vector of claim 24 wherein said mature protein is a PspAlipoprotein of a strain of S. pneumoniae.
 27. The expression vector ofclaim 24 wherein said mature protein is a UreA protein of a strain of H.pylori.
 28. The expression vector of claim 24 wherein said matureprotein is a UreB protein of a strain of H. pylori.
 29. An expressionvector containing the hybrid nucleic acid molecule of claim 18 undercontrol of a promoter for expression of said OspC lipoprotein.
 30. Anexpression vector containing the hybrid nucleic acid molecule of claim21 under control of a promoter for expression of said PspA protein. 31.An expression vector containing the hybrid nucleic acid molecule ofclaim 22 under control of a promoter for expression of said UreAprotein.
 32. An expression vector containing the hybrid nucleic acidmolecule of claim 23 under control of a promoter for expression of saidUreB protein.
 33. A method for forming a recombinant protein, whichcomprises: incorporating the expression vector of claim 24 into a hostorganism; and effecting expression of said mature protein from the hostorganism.
 34. The method of claim 33 wherein said mature protein is anOspC lipoprotein of a Borrelia species.
 35. The method of claim 34wherein said host organism is E. coli.
 36. The method of claim 33wherein said mature protein is a PspA protein of a strain of S.pneumoniae.
 37. The method of claim 36 wherein said host organism is E.coli.
 38. The method of claim 33 wherein said mature protein is a UreAprotein of a strain of H. pylori.
 39. The method of claim 38, whereinsaid host organism is E. coli.
 40. The method of claim 33 wherein saidmature protein is a UreB protein of a strain of H. pylori.
 41. Themethod of claim 40 wherein said host organism is E. coli.
 42. A methodfor forming recombinant ospC lipoprotein, which comprises: incorporatingthe expression vector of claim 29 into a host organism; and effectingexpression of said OspC lipoprotein from the host organism.
 43. Themethod of claim 42 wherein said host organism is E. coli.
 44. A methodfor forming recombinant PspA lipoprotein, which comprises: incorporatingthe expression vector of claim 30 into a host organism; and effectingexpression of said PspA lipoprotein from the host organism.
 45. Themethod of claim 44 wherein said host organism is E. coli.
 46. A methodfor forming recombinant UreA lipoprotein, which comprises: incorporatingthe expression vector of claim 31 into a host organism; and effectingexpression of said UreA lipoprotein from the host organism.
 47. Themethod of claim 46 wherein said host organism is E. coli.
 48. A methodfor forming recombinant UreB lipoprotein, which comprises: incorporatingthe expression vector of claim 32 into a host organism; and effectingexpression of said UreA lipoprotein from the host organism.
 49. Themethod of claim 48 wherein said host organism is E. coli.
 50. A processfor the production of a recombinant lipoprotein, which comprises:constructing a hybrid nucleic acid molecule comprising a first nucleicacid sequence encoding a signal sequence of a lipoprotein and a secondnucleic acid sequence encoding a mature protein, or fragment thereof,which is heterologous to the lipoprotein encoded by said first nucleicacid, said second nucleic acid sequence being contiguous with said firstsequence; forming an expression vector containing said hybrid nucleicacid molecule under control of a promoter for expression of said matureprotein; incorporating said expression vector into a host organism;effecting expression of said recombinant lipoprotein by said hostorganism; lysing the cells of the host organism; treating the lysedcells with a surfactant which selectively solubilizes said recombinantlipoprotein in preference to bacterial and other proteins and which isable to effect phase separation of a detergent phase under mildconditions; effecting phase separation at a detergent phase containingsolubilized recombinant lipoprotein, an aqueous phase containingbacterial and other proteins and a solid phase containing cell residue;separating and recovering said detergent phase from said solid phase andsaid aqueous phase; contacting said detergent phase with a firstchromatographic column under conditions which result in binding ofprotein other than said recombinant lipoprotein to said column toprovide a flow-through from said first chromatographic column containingthe recombinant lipoprotein and recovering said flow-through from saidfirst chromatographic column; contacting the flow-through from saidfirst chromatographic column with a second chromatographic column underconditions which result in binding of the recombinant lipoprotein to thesecond chromatographic column in preference to contaminant proteins andlipopolysaccharides which pass through said second chromatographiccolumn; eluting said recombinant lipoprotein from said secondchromatographic column to provide an eluant containing said recombinantlipoprotein substantially free from lipopolysaccharide and contaminantproteins; and recovering said eluant.
 51. The process of claim 50wherein said signal sequence is the signal sequence of an OspA proteinof a Borrelia species.
 52. The process of claim 51 wherein said firstnucleic acid sequence and said second nucleic acid sequence are coupledin a translational open reading frame relationship.
 53. The process ofclaim 52 wherein said surfactant is TRITON™ X-114.
 54. The process ofclaim 53 wherein said treating of lysed cells is effected at atemperature of about 0° C. to about 10° C., the resulting mixture istreated to a mildly elevated temperature of about 35° C. to about 40° C.to effect separation of said detergent phase, and said detergent phaseis separated from said aqueous phase and said solid phase bycentrifugation.
 55. The process of claim 52 wherein said mature proteinis an OspC lipoprotein of a Borrelia species.
 56. The process of claim55 wherein said first chromatographic column is further contacted with abuffer medium at a pH to provide liquid containing the recombinant ospClipoprotein from the first chromatographic column while the otherproteins are retained on the first chromatographic column and theflow-through from the further contact is collected and combined withthat from the first contacting step on said first chromatographic columnand the combined flow-through is contacted with said secondchromatographic column.
 57. The process of claim 52 wherein said matureprotein is a PspA protein of a strain of S. pneumoniae.
 58. The processof claim 57, wherein said first chromatographic column is furthercontacted with a buffer medium at a pH to provide liquid containing therecombinant PspA lipoprotein from the first chromatographic column whilethe other proteins are retained on the first chromatographic column andthe flow-through from the further contact is collected and combined withthat from the first contacting step on said first chromatographic columnand the combined flow-through is contacted with said secondchromatographic column.
 59. The process of claim 52 wherein said matureprotein is a UreA protein of a strain of H. pylori.
 60. The process ofclaim 59 wherein said first chromatographic column is further contactedwith a buffer medium at a pH to provide liquid containing therecombinant UreA lipoprotein from the first chromatographic column whilethe other proteins are retained on the first chromatographic column andthe flow-through from the further contact is collected and combined withthat from the first contacting step on said first chromatographic columnand the combined flow-through is contacted with said secondchromatographic column.
 61. The process of claim 52 wherein said matureprotein is a UreB protein of a strain of H. pylori.
 62. The process ofclaim 61 wherein said first chromatographic column is further contactedwith a buffer medium at a pH to provide liquid containing therecombinant UreB lipoprotein from the first chromatographic column whilethe other proteins are retained on the first chromatographic column andthe flow-through from the further contact is collected and combined withthat from the first contacting step on said first chromatographic columnand the combined flow-through is contacted with said secondchromatographic column.
 63. The process of claim 52 wherein said hostorganism lysis is effected by freezing and thawing the host organism.64. Recombinantly-produced, isolated and purified lipoprotein producedby the process of claim
 50. 65. Recombinantly-produced, isolated andpurified ospC lipoprotein of a Borrelia strain having a purity of atleast about 80% and substantially free from contaminant proteins andlipopolysaccharides.
 66. Recombinantly-produced, isolated and purifiedlipidated PspA protein of a strain of S. pneumoniae having a purity ofat least about 50% and substantially free from contaminant proteins andlipopblysaccharides.
 67. Recombinantly-produced, isolated and purifiedlipidated UreA protein of a strain of H. pylori having a purity of atleast about 80% and substantially free from contaminant proteins andlipopolysaccharides.
 68. Recombinantly-produced, isolated and purifiedlipidated UreB protein of a strain of H. pylori having a purity of atleast about 80% and substantially free from contaminant proteins andlipopolysaccharides.
 69. A lipoprotein fusion vector PLF100 having ATCCAccession No.
 69750. 70. A method for inducing an immunological responsein a human or animal comprising the step of administering to said humanor animal a composition comprising the lipoprotein of claim 64 or 65.71. A composition for inducing an immunological response comprising thelipoprotein of claim 64 or
 65. 72. A method for inducing animmunological response in a human or animal comprising the step ofadministering to said human or animal a composition comprising thelipidated PspA protein of claim 64 or
 66. 73. A composition for inducingan immunological response comprising the lipidated PspA protein of claim64 or
 66. 74. A method for inducing an immunological response in a humanor animal comprising the step of administering to said human or animal acomposition comprising the lipidated UreA protein of claim
 67. 75. Acomposition for inducing an immunological response comprising thelipidated UreA protein of claim
 67. 76. A method for inducing animmunological response in a human or animal comprising the step ofadministering to said human or animal a composition comprising thelipidated UreB protein of claim
 68. 77. A composition for inducing animmunological response comprising the lipidated UreB protein of claim68.
 78. A method for enhancing the immunogenicity of a protein,comprising the steps of: forming a hybrid nucleic acid moleculecomprising a first nucleic acid sequence encoding a signal sequence of alipoprotein and a second nucleic acid sequence encoding a matureprotein, or fragment thereof, which is heterologous to the lipoproteinencoded by said first nucleic acid, said first nucleic acid sequencebeing contiguous with said second nucleic acid sequence; incorporatingsaid hybrid nucleic acid molecule into an expression vector; effectingexpression vector into a host organism; and effecting expression of saidmature protein from said host organism.